resulting in a wide displacement of the PsR
domain. As the pool of free KaiA dimers is depleted, KaiC switches back to autodephosphorylation activity. Complete dephosphorylation of
KaiC results in dissociation of the KaiCBA complex by loss of KaiA2B1 subcomplexes, thereby
completing one cycle of the oscillator. In cyanobacterial cells, KaiC and KaiB are produced from
the same operon and in 10- to 100-fold excess to
KaiA (28). The high excess of KaiCB over free
KaiA could promote efficient sequestration of
KaiA in vivo. The model presented here can thus
serve as a framework to better understand the
circadian clock in cyanobacterial cells.

We thank J. Andres and M. Yazdanyar for help with protein
expression, O. Mihalache for help with sample preparation, F. Beck
and A. Aufderheide for assistance with image processing, and
C. Benda for help with PHENIX software. This work was supported
by the Netherlands Organisation for Scientific Research (NWO)

Roadmap Initiative Proteins@Work grant 184.032.201 to A.J.R.H.,
the European Union Seventh Framework Programme ManiFold
grant 317371 to A.J.R.H. and P.L., and the German Research
Foundation grant GRK1721 to F.F., and grants AX 84/1-3 and EXC
1028 to A. W., N.S., and I.M.A.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/355/6330/1181/suppl/DC1

Materials and Methods
Figs. S1 to S15

Tables S1 to S4

References (29–51)

Data S1 to S3

10 June 2016; accepted 13 February 2017

10.1126/science.aag3218

AGING

Aggregation of the Whi3 protein, notloss of heterochromatin, causessterility in old yeast cells

Gavin Schlissel,1 Marek K. Krzyzanowski,2 Fabrice Caudron,2,3

Yves Barral,2† Jasper Rine1†

In yeast, heterochromatin silencing is reported to decline in aging mother cells, causing sterility
in old cells. This process is thought to reflect a decrease in the activity of the NAD+ (oxidized
nicotinamide adenine dinucleotide)–dependent deacetylase Sir2. We tested whether Sir2
becomes nonfunctional gradually or precipitously during aging. Unexpectedly, silencing of the
heterochromatic HML and HMR loci was not lost during aging. Old cells could initiate a mating
response; however, they were less sensitive to mating pheromone than were young cells
because of age-dependent aggregation of Whi3, an RNA-binding protein controlling S-phase
entry. Removing the polyglutamine domain of Whi3 restored the pheromone sensitivity of old
cells. We propose that aging phenotypes previously attributed to loss of heterochromatin
silencing are instead caused by aggregation of the Whi3 cell cycle regulator.

Budding yeast divide asymmetrically, and each yeast mother cell produces a finite number of daughter cells in its lifetime. This process—yeast replicative aging—has been studied for insights into aging more
broadly, because the processes that underlie aging
in yeast might be related to factors that underlie
aging in other asymmetrically dividing cells (1).

In Saccharomyces cerevisiae, haploid mothercells lose the ability to mate as they age (2). It hasbeen proposed that old mother cells fail to mateas a consequence of a decline in Sir2 function,which would cause loss of heterochromatic genesilencing of the auxiliary mating-type loci HMLand HMR (3). Loss of silencing at HML and HMRin old cells has been attributed to the redistribu-tion of Sir proteins to the nucleolus and to a de-crease in available Sir2 (4–6). Furthermore, oldcells may be either limited for the Sir2 substratenicotinamide adenine dinucleotide (oxidized form;

NAD+) or exposed to high concentrations of
nicotinamide (NAM), an inhibitor of Sir2, resulting in the inactivation of Sir2 in old cells
and thus sterility (7–9).

We characterized transcriptional repression bySir2 by testing whether transient loss-of-silencingevents at HML might precede the complete lossof silencing attributed to the oldest cells. To studysilencing at HML in a yeast mother cell, we mon-itored pedigrees of haploid cells carrying a Cre-based silencing reporter (10). The reporter uses aCre recombinase gene inserted in place of HMLa2and a fluorescent reporter inserted at a euchromaticlocus elsewhere in the genome (Fig. 1A). Loss ofsilencing at hmla2D::CRE induces a permanentand heritable switch from expressing red fluores-cent protein (RFP) to expressing green fluorescentprotein (GFP) (Fig. 1A), and the sensitivity of theCre reporter approaches the sensitivity of single-molecule RNA fluorescent in situ hybridization(10). We manually separated daughter cells fromtheir mothers to analyze pedigrees in two commonstrain backgrounds, S288c and W303, and observedno loss-of-silencing events in dozens of pedigreesof haploids, diploids, and hybrids (Fig. 1B).To measure the frequency of silencing loss as afunction of a cell’s life span, we extended thepedigree analysis by using a microfluidic devicethat traps mother cells and separates their buds.We analyzed more than 1500 yeast pedigrees atsingle-cell resolution and observed 13 loss-of-silencing events (Fig. 1C and movie S1). Further-more, we found that a cell’s age did not affect itsability to maintain silencing of HML, and theoverwhelming majority of yeast mother cellsstopped dividing without even a transient loss ofsilencing (Fig. 1D). As a control, when Sir2 ac-tivity in old cells was inhibited by addition ofNAM, all surviving cells lost silencing, suggestingthat old cells did not accumulate NAM in amountsthat inactivate Sir2 (Fig. 1E and movie S2). Similarresults were obtained by analyzing a GFP geneinserted in place of HMLa and using an alter-native microfluidic design, indicating that theobservation was independent of the reporter andthe microfluidic setup used (fig. S1). Previousstudies have shown that Sir2 protein levels de-crease in cells that are more than seven genera-tions old; however, we found no evidence of adecrease in Sir2 activity at HML (4). It is possiblethat a decrease in Sir2 levels in old cells could affectother Sir2 complexes, including the nucleolar RENT